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Xenopus laevis; cytoplasmic retention; mi-erl; transcription factor; nuclear translocation
Abstract:
Xmi-er1 is a fibroblast growth factor regulated immediate-early gene that is activated during mesoderm induction in Xenopus embryonic explants. This gene encodes a nuclear protein with potent transcriptional regulator activity and overexpression of XMI-ER1 in Xenopus embryos inhibits mesoderm induction and leads to truncations along the anteroposterior axis. We showed previously that XMI-ER1 is retained in the cytoplasm during cleavage stages and only begins to appear in the nucleus at midblastula. Such developmentally regulated nuclear translocation may represent an important mechanism for regulating XMI-ER1 activity in the early embryo. Here, we investigate different mechanisms that might control nuclear translocation of XMI-ER1. Using α-amanitin to inhibit transcription, we show that nuclear localization is not dependent on zygotic transcription. Nor is it the result of a developmentally regulated import pathway, as the XMI-ER1 nuclear localization signal (NLS) fused to beta-galactosidase (βgal) was able to direct nuclear translocation prior to mid-blastula. Fusion of an additional, heterologous NLS to the N-terminus of XMI-ER1 was not sufficient to overcome cytoplasmic retention, indicating that retention does not involve NLS masking, but rather binding to a cytoplasmic anchor. The anchoring molecule is not an RNA, as microinjection of RNase A did not affect the timing of nuclear translocation.Western blot analysis using antibodies that recognize phosphorylated residues revealed that, while XMI-ER1 is not itself phosphorylated, it is associated with two differentially phosphorylated proteins, suggesting that the anchoring mechanism may involve interaction with a cytoplasmic protein(s). A series of XMI-ER1 deletion mutants was utilized to map the putative retention domain. Our analysis revealed that amino acids 144–175, containing the fourth acidic stretch of the acidic activation domain, are required for retention. These results suggest that XMI-ER1 is retained in the cytoplasm of the early embryo by interaction of the region containing amino acids 144–175 with a cytoplasmic anchor.
